PepFect14, a Versatile Cell-Penetrating Peptide

PepFect14, a Versatile Cell- Penetrating Peptide

Maxime Gestin

Maxime Gestin PepFect14, a Versatile Cell-Penetrating Peptide

Department of Biochemistry and Biophysics

ISBN 978-91-7911-110-6

Maxime Gestin

MG graduated in a B.Sc. and a M.Sc.

in Chemistry at Université de Bretagne Occidentale after two years in the Graduate School of Chemistry, Biology and Physics of Bordeaux. MG started his PhD studies at

Stockholms Universitet in 2015.

Cell-penetrating peptides have been discovered almost three decades ago and there are, nowadays, thousands of available sequences. They offer multiple applications in the field of drug delivery as they are able to carry therapeutic macromolecules across the plasma membrane.

Throughout the years, new sequences have been developed and designed to achieve new applications such as specificity for certain kinds of cargoes, intrinsic therapeutic effects and targeted delivery.

In this thesis, we focused on a single most promising cell-penetrating peptide named PepFect14 and aimed at reaching a better understanding of the factors involved in the cellular uptake and at discovering and developing new applications for PepFect14 in order to broaden its potential.

Overall, this thesis summarizes our effort to develop and bring to their full potential already existing cell-penetrating peptides instead of developing new sequences for each new application.

PepFect14, a Versatile Cell-Penetrating Peptide

Maxime Gestin

Academic dissertation for the Degree of Doctor of Philosophy in Neurochemistry with Molecular Neurobiology at Stockholm University to be publicly defended on Wednesday 10 June 2020 at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B.

Abstract

Cell-penetrating peptides have been discovered almost three decades ago and there are, nowadays, thousands of available sequences. They offer multiple applications in the field of drug delivery as they are able to carry therapeutic macromolecules across the plasma membrane. Throughout the years, new sequences have been developed and designed to achieve new applications such as specificity for certain kinds of cargoes, intrinsic therapeutic effects and targeted delivery.

In this thesis, we focused on a single most promising cell-penetrating peptide named PepFect14 and aimed at reaching a better understanding of the factors involved in the cellular uptake through paper I and paper II. Notably, in paper I we screened a library of small molecule drugs that influences signaling pathways and discovered that three drugs had an unreported influence on endocytosis. In paper II, After performing an RNA sequencing on cells treated with PepFect14, we demonstrated the involvement of autophagy in the intracellular trafficking of the cell-penetrating peptide. A second aim of this thesis, covered in paper III and paper IV, was to discover new applications for PepFect14 in order to broaden its potential. In paper III, we successfully used PepFect14 to mediate the intracellular delivery of heat shock protein 70kDa.

This was the first protein delivery assisted by PepFect14. In paper IV, PepFect14 was covalently fused to mtCPP1, a cell- penetrating peptide that targets mitochondria and reduce the level of reactive oxygen species. The constructs showed the ability to keep the properties of both peptides and achieved a mitochondria-targeted antisense therapy.

Overall, this thesis summarizes our effort to develop and bring to their full potential already existing cell-penetrating peptides instead of developing new sequences for each new application.

Keywords: Cell-Penetrating Peptides, PepFect14, transfection, signaling mechanisms, intracellular targeting.

Stockholm 2020

http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-180948

ISBN 978-91-7911-110-6 ISBN 978-91-7911-111-3

Department of Biochemistry and Biophysics

Stockholm University, 106 91 Stockholm

PEPFECT14, A VERSATILE CELL-PENETRATING PEPTIDE

Maxime Gestin

PepFect14, a Versatile Cell- Penetrating Peptide

Maxime Gestin

©Maxime Gestin, Stockholm University 2020 ISBN print 978-91-7911-110-6

ISBN PDF 978-91-7911-111-3

"La science, mon garçon, est faite d’erreurs, mais d’erreurs qu’il est bon de commettre,

car elles mènent peu à peu à la vérité."

       Jules Verne

A ^bstract

Cell-penetrating peptides have been discovered almost three decades ago and there are, nowadays, thousands of available sequences. They offer multi- ple applications in the field of drug delivery as they are able to carry therapeu- tic macromolecules across the plasma membrane. Throughout the years, new sequences have been developed and designed to achieve new applications such as specificity for certain kinds of cargoes, intrinsic therapeutic effects and targeted delivery.

In this thesis, we focused on a single most promising cell-penetrating pep- tide named PepFect14 and aimed at reaching a better understanding of the factors involved in the cellular uptake through paper I and paper II. Notably, in paper I we screened a library of small molecule drugs that influence signal- ing pathways and discovered that three drugs had an unreported influence on endocytosis. In paper II, After performing an RNA sequencing on cells treated with PepFect14, we demonstrated the involvement of autophagy in the intra- cellular trafficking of the cell-penetrating peptide. A second aim of this thesis, covered in paper III and paper IV, was to discover new applications for Pep- Fect14 in order to broaden its potential. In paper III, we successfully used PepFect14 to mediate the intracellular delivery of heat shock protein 70kDa.

This was the first protein delivery assisted by PepFect14. In paper IV, Pep- Fect14 was covalently fused to mtCPP1, a cell-penetrating peptide that targets mitochondria and reduces the level of reactive oxygen species. The constructs showed the ability to keep the properties of both peptides and achieved a mi- tochondria-targeted antisense therapy.

Overall, this thesis summarizes our effort to develop and bring to their full potential already existing cell-penetrating peptides instead of developing new sequences for each new application.

P opulärvetenskaplig Sammanfattning

Under senare år har användningen av genterapi, det vill säga terapier som riktar sig mot våra cellers DNA, utvecklats mycket. Denna utveckling har lett till nya problem som det vetenskapliga samfundet ska lösa. Faktum är att ma- joriteten av läkemedlen använder sig av små molekyler för att komma in i cellerna medan genterapi använder molekyler av större storlekar som kallas oligonukleotider. Oligonukleotider kan inte komma in i cellerna själva, vilket har begränsat deras användning under flera decennier. En mängd system har utvecklats för att underlätta upptag av oligonukleotider i celler.

Denna avhandling passar in i detta forskningsområde och specialiserar sig på ett av dessa system som kallas cellpenetrerande peptider. Cellpenetrerande peptider är molekyler av medelstor storlek som har kapacitet att passera ge- nom cellulära barriärer och kan transportera terapeutiska molekyler av större storlek. De studier som presenteras i denna avhandling fokuserar på en speci- fik cellpenetrerande peptid som har utvecklats i vår forskargrupp, nämligen PepFect14. Tusentals andra cellpenetrerande peptider existerar, var och en med specifika egenskaper och nya utvecklas varje år. PepFect14 är dock en mycket bra kandidat som redan har visat stor potential inom genterapi.

I den här avhandlingen är vårt första mål att bättre förstå PepFect14s mek- anismer för att låta den nå sin fulla terapeutiska potential. I artikel I upptäckte vi tre molekyler som kan användas parallellt med PepFect14 och som har ka- pacitet att tredubbla effekterna av genterapibehandlingen. I artikel II tittade vi

på hur celler svarar på upptag av PepFect14 och visade att ett naturligt system för återvinning av celler, autofagi, utlöstes av behandlingen.

Det andra målet med vårt arbete var att utöka användningsområden av PepFect14. Hittills har denna cellpenetrerande peptid endast använts för gen- terapi och det är därför som vi i artikel III har visat på förmågan hos PepFect14 att hjälpa vid införandet av terapeutiska proteiner i celler. Proteiner är en an- nan klass av biologiska molekyler vars användning utgör ett mindre invasivt alternativ än oligonukleotider. Användningen av proteinbaserade terapier har emellertid samma begränsningar som oligonukleotider eftersom de inte heller kan penetrera cellerna ensamma. Vi har således demonstrerat att PepFect14 inte bara har kapacitet att tillåta oligonukleotider att upptas i celler utan även proteiner. Dessa två applikationer gör PepFect14 till en peptid med stor pot- ential. Slutligen, i artiklarna IV, visade vi ännu en ny applikation för PepFect14. Vissa behandlingar behöver nå en specifikt position i cellerna. När det gäller genterapi är främsta målet mitokondrierna. Vi har fusionerat struk- turen för PepFect14 med en annan cellpenetrerande peptid som kallas mtCPP1. MtCPP1 har förmågan att kunna riktas direkt mot mitokondrier men de kan inte transportera oligonukleotider. Resultatet av fusionen av dessa två peptider, kallade mitFects, kombinerade egenskaperna hos varje peptid och tillät oss att genomföra genterapi riktad direkt mot mitokondrierna.

Alla dessa resultat visar vikten av att utveckla befintliga cellpenetrerande peptider för att ge dem tillgång till deras fulla potential. Genom de fyra artik- larna samlade i denna avhandling har vi visat att PepFect14 är en mångsidig cellpenetrerande peptid, som kan anpassas för att vara effektiv inom flera te- rapeutiska områden.

R ésumé Vulgarisé

Depuis quelques années, l’utilisation de la thérapie génique, c’est à dire des thérapies qui visent l’ADN de nos cellules, s’est largement développée. Cette évolution a amené de nouvelles problématiques à résoudre par la communauté scientifique. En effet, la majorité des médicaments utilisés jusqu’alors était des molécules de petites tailles qui pouvaient rentrer dans les cellules sans grandes difficultés alors que la thérapie génique utilise des molécules de grandes tailles appelées des oligonucléotides. Les oligonucléotides ne peuvent pas entrer dans les cellules d’eux-mêmes, ce qui a limité leur utilisation pen- dant plusieurs décennies. Une multitude de systèmes a été développée pour assister leur entrée dans les cellules.

Cette thèse prend part à ce champs de recherche et se concentre spéciale- ment sur un de ces systèmes appelé les peptides pénétrants. Les peptides pé- nétrants sont des molécules de moyennes tailles qui ont la capacité de passer au travers des barrières cellulaires et de transporter avec eux des molécules thérapeutiques de grandes tailles. Les différentes études présentées dans cette thèse se concentrent sur un peptide pénétrant développé dans notre groupe de recherche dénommé PepFect14. Des milliers d’autres peptides pénétrants existent, chacun avec des propriétés particulières et de nouveaux sont déve- loppés chaque année. Cependant, PepFect14 est un très bon candidat qui a déjà montré un fort potentiel dans la thérapie génique.

Dans cette thèse, notre premier but est de mieux comprendre les méthodes d’action de PepFect14 afin de lui permettre d’atteindre son plein potentiel thé- rapeutique. En particulier, dans l’article I, nous avons découvert trois molé- cules qui peuvent être utilisées en parallèle de PepFect14 et qui ont la capacité de tripler les effets du traitement. Dans l’article II, nous nous sommes intéres- sés aux réactions des cellules à notre traitement et nous avons démontré qu’un système naturel de recyclage cellulaire, l’autophagie, était déclenché en ré- ponse à PepFect14.

Le second but de notre travail était d’étendre le champs d’action de PepFect14. Jusqu’à présent ce peptide pénétrant n’était utilisé que pour de la thérapie génique et c’est pourquoi dans l’article III, nous avons montré la ca- pacité de PepFect14 à assister l’entrée dans les cellules de protéines thérapeu- tiques. Les protéines sont une autre classe de molécules biologiques et leur utilisation présente une alternative moins invasive que les oligonucléotides.

Cependant, l’utilisation de thérapies basées sur des protéines présente les même limitations que les oligonucléotides, étant donné qu’elles ne peuvent pas non plus pénétrer les cellules seules. Nous avons ici montré que PepFect14 avait non seulement la capacité de permettre à des oligonucléotides de rentrer dans les cellules mais aussi à des protéines. Ces deux applications font déjà de PepFect14 un peptide portant un grand potentiel. Enfin, dans l’articles IV, nous avons montré une nouvelle application pour PepFect14. En effet, certains traitements ont besoin d’être amenés à une cible particulière dans les cellules.

En ce qui concerne la thérapie génique, des cibles de choix sont les mitochon- dries. Nous avons fusionné la structure de PepFect14 avec un autre peptide pénétrant appelé mtCPP1 qui a la capacité de cibler les mitochondries mais ne peut pas transporter d’oligonucléotides. Le résultat de la fusion de ces deux peptides, appelé mitFects, a non seulement conservé mais aussi combiné les propriétés de chaque parties et nous a permis de réaliser une thérapie génique ciblée sur les mitochondries.

L’ensemble de ces résultats montre l’importance du développement des peptides pénétrants existant afin de leur donner accès à leur plein potentiel. A travers les quatre articles rassemblés cette thèse, nous avons démontré que PepFect14 est un peptide pénétrant versatile, qui peut être adapté pour être efficace dans plusieurs domaines de traitement thérapeutique.

L ist of Publications

This thesis is based on the following four papers. In the text, the papers will be referred to as Paper I, II, III and IV.

I. Gestin M., Helmfors H., Falato L., Lorenzon N., Michalakis F. I., Langel Ü. Effect of Small Molecule Signaling in PepFect14 Trans- fection. PLoS ONE 15 (1), e0228189 (2020).

II. Dowaidar M., Gestin M., Cerrato C. P., Jafferali M. H., Margus H., Kivistik P. A., Ezzat K., Hallberg E., Pooga M., Hällbrink M., Langel Ü. Role of Autophagy in Cell-Penetrating Peptide Trans- fection Model. Scientific Reports 7 (1), 12635 (2017).

III. Gestin M., Falato L., Ciccarelli M., Andréasson C., Langel Ü.

Transfection of Heat Shock Protein 70 kDa (HSP70). Manuscript.

IV. Cerrato C. P., Kivijärvi T., Tozzi R., Lehto T., Gestin M., Langel Ü. Intracellular Delivery of Therapeutic Antisense Oligonucleo- tides Targeting mRNA Coding Mitochondrial Proteins by Cell- Penetrating Peptides. Manuscript.

A dditional Publications

Publications not included in this thesis:

V. Kurrikoff K., Gestin M., Langel Ü. Recent in vivo advances in cell-penetrating peptide-assisted drug delivery. Expert Opinion on Drug Delivery 13 (3), 373-387 (2016).

VI. Gestin M., Dowaidar M., Langel Ü. Uptake mechanism of cell- penetrating peptides. Advances in Experimental Medicine and Bi- ology 1030, 255-264 (2017).

VII. Lorenzon N., Gestin M., Langel Ü. Mimicry of dopamine recep- tor 1 signaling with cell-penetrating peptides. Manuscript ac- cepted for publication in International Journal of Peptide Research and Therapeutics.

VIII. Falato L., Gestin M., Langel Ü. Cell-penetrating peptides deliver- ing siRNAs: an overview. Manuscript.

IX. Carreras-Badosa G., Maslovskaja J., VaherH., UrgardE., Padari K., PeriyasamyK., TserelL, GestinM., KisandK., ArukuuskP., LouC., LangelÜ., Wengel J.,Pooga M., RebaneA. NickFect type of cell-penetrating peptides present enhanced efficiency for mi- croRNA-146a delivery into dendritic cells and during skin inflam- mation. Manuscript.

A bbreviations

Antp Drosophilia atennapedia homeobox protein ASO Antisense oligonucleotide

ADP Adenosine diphosphate ATP Adenosine triphosphate BHM Bormiski hamster melanoma cDNA Complementary DNA

CME Clathrin-mediated endocytosis COXII Cytochrome c oxidase subunit II CPP Cell-penetrating peptide

CRISPR/Cas9 Clustered regularly interspaced short palindromic re- peats/CRISPR asso-ciated nuclease 9

DIC N,N'-diisopropylcarbodiimide DLS Dynamic light scattering

DMEM Dubelco’s modified Eagle medium ESI-TOF Electrospray ionization – Time of flight FBS Fetal bovine serum

Fmoc 9-fluorenylmethoxycarbonyl HIV-1 Human immunodeficiency virus 1 HRH3 Histamine receptor H 3

HSP70 Heat shock protein 70kDa IMM Inner mitochondrial membrane IPA Ingenuity Pathway Analysis mGluR5 Metabotropic glutamate receptor 5 MPEP 2-Methyl-6-(phenylethynyl)-pyridine MR Molar ratio

mRNA messenger RNA mtDNA Mitochondrial DNA

NBD Adenine nucleotide binding domain

OMM Outer mitochondrial membrane ON Oligonucleotide

OXPHOS Oxidative phosphorylation ROS Reactive oxygen species

RP-HPLC Revers phase high performance liquid chromatography SBD Substrate binding domain

SCARA Scavenger receptor type A SCO Splice correcting oligonucleotide siRNA Small interfering RNA

SPPS Solid phase peptide synthesis TAT Trans-activator of transcription TEM Transmission electron microscopy TFA Trifluoroacetic acid

TMRE Tetramethylrhodamine ethyl ester UCP2 Mitochondrial uncoupling protein 2

UHPLC-MS Ultra-high performance liquid chromatography – Mass spectrometry

DYm Mitochondrial membrane potential

C ^ontents

Abstract ... i Populärvetenskaplig Sammanfattning ... iii Résumé Vulgarisé ... v List of Publications ... ix Additional Publications ... x Abbreviations ... xi Introduction ... 1

Cell-penetrating peptides ... 1 History of cell-penetrating peptides ... 2 PepFect 14 ... 4 Uptake mechanisms and cellular trafficking ... 6 Signaling Pathways ... 10 Autophagy ... 11 Delivery of therapeutics ... 15 Gene therapy ... 16 Antisense therapy ... 17 Targeted delivery to intracellular organelles ... 18 Mitochondria as a therapeutic target ... 19 Protein delivery ... 20 Heat shock protein 70kDa ... 21

Aims ... 25

Paper I ... 26 Paper II ... 26 Paper III ... 26 Paper IV ... 27

Methods ... 29

Solid-Phase Peptide Synthesis ... 29 Cell cultures ... 33

Luciferase assay ... 34 Splice correcting assay ... 35 RNA analysis ... 37 RNA extraction ... 37 RNA sequencing ... 37 Real time quantitative polymerase chain reaction ... 38 Western Blot analysis ... 39 Microscopy ... 41 Fluorescent Microscopy ... 41 Transmission electron microscopy ... 42 Tryptophan fluorescence extinction ... 43 Dynamic light scattering ... 44 Mitochondrial activity ... 45 Determination of mitochondrial potential ... 45 Determination of reactive oxygen species levels ... 45 Cell proliferation assay ... 46

Results and Discussions ... 47

Paper I ... 47 Paper II ... 49 Paper III ... 52 Paper IV ... 54

Conclusions and Future Outlook ... 57

Paper I and paper II ... 57 Paper III and paper IV ... 58

Acknowledgments ... 61 References ... 63

I ntroduction

Cell-penetrating peptides

Cell-penetrating peptides (CPPs) are a class of peptide that are able to pen- etrate cells as their name suggests. In other words, CPPs have the ability to overcome cellular barrier such as biological membranes and translocate into cells without toxic effects^1,2. Most of them can be conjugated covalently or non-covalently to cargo molecules and can carry them across the cell mem- brane into the cytosol, the nucleus or other sites of therapeutic interest³. The cargoes can be of various natures. CPPs have been used as vehicles for fluo- rescent probes^4,5, contrast agents for magnetic resonance imaging⁶, as well as bioactive small molecules, proteins^7,8 or oligonucleotides^9–12. Some CPPs even have an intrinsic therapeutic effect in addition to their penetration abili- ties^13,14. This large range of applications makes CPPs a formidable tool for the delivery of therapeutics directly inside the cell. A summary of the possible cargoes carried by CPPs is given in Figure 1. Furthermore, the sequence of a peptide can be optimized through a rational functionalization allowing the binding of virtually all cargoes. Even though the CPP family has broadened exponentially since their discovery they all present some common features;

they are usually short peptides with less than thirty amino acids and most are cationic and/or amphipathic¹⁵ (although some are anionic¹⁶). Cationic and am- phipathic CPPs offer the opportunity to bind to negatively charged oligonu- cleotides (ONs) in a non-covalent way, the opposite charges assisting the for- mation of a complex¹⁷.

Figure 1. Applications of CPPs as molecular delivery vehicles for a variety of drugs, nucleic acids, proteins, therapeutics, and imaging agents. Reprinted with permission¹⁸.

History of cell-penetrating peptides

The first identified protein domain with transduction abilities was discov- ered simultaneously by two different research groups in 1988^19,20. In both Frankel and Pabo²⁰ and Green and Loewenstein¹⁹ studies, the trans-activator of transcription (TAT) of human immunodeficiency virus 1 (HIV-1) was de- scribed as able to cross cellular membranes and to be internalized by cells in vitro. Before the discovery of TAT, it was thought that peptides and proteins were not able to cross plasma membranes. The second protein transduction domain that was identified also came from a natural protein and was found in Drosophilia atennapedia homeobox protein (Antp)²¹. Further studies aimed at

reducing the length of the sequence of these proteins to find the parts respon- sible for the cell-penetration^22,23. TAT sequence got reduced to 13 amino ac- ids²³ and Antp was truncated to give a 16 amino acids peptide called pene- tratin²². The concept of CPPs was introduced by Ülo Langel’s group in 1998 when transportan, a chimeric peptide that was not a protein transduction do- main, was designed and shown to penetrate cells²⁴. Also in the end of the 1990s Prof. Dowdy’s group published a successful transduction of TAT fused with a protein both in vitro and in vivo. The proteins remained active within their new host cells^25,26.

Within almost three decades, the number of CPPs increased rapidly to reach around two thousands different sequences in 2016²⁷ including natural protein derived peptides such as pVec²⁸, chimeric peptide like transportan²⁴ and Pep- Fect14²⁹ and synthetic peptide such as polyarginines³⁰ and MAP³¹ (model am- phipathic peptide). Most of the available CPPs have a linear sequence even though some occurrence of cyclic CPPs^32,33 and branched sequences³⁴ can be found in literature. Table 1 presents examples of commonly studied CPPs that have shown an efficient uptake into cells.

Table 1. Examples of commonly studied CPPs

Peptide Sequence Reference

Protein derived

TAT GRKKRRQRRRPPQ 23

Penetratin RQIKIWFQNRRMKWKK 22

pVEC LLIILRRRIRKQAHAHSK-NH2 28

Chimeric

Transportan GWTLNSAGYLLGKINLKALAALAKKIL-NH2 24

TP10 AGYLLGKINLKALAALAKKIL-NH2 36

Synthetic

Polyarginine Rn (6 < n < 12) 30

MAP KLALKLALKALKAALKLA-NH2 31

The sequences in table 1 are categorized according to their origin but other classification systems exist. One can also categorize CPPs according to their physico-chemical properties. In this case the most used sub-categories are cat- ionic sequences, amphipathic peptides and hydrophobic peptides³⁵. Most pep- tides are cationic at physiological pH. The positive charges of these peptides usually come from lysine and arginine residues. Amphipathic CPPs combine positively charged and hydrophobic residues and the amphipacity can either be due the primary structure of the peptides or their secondary structure. Hy- drophobic peptides possess charged residues separated from hydrophobic parts on their amino acids backbone.

PepFect 14

PepFect14 (PF14) is a peptide developed in Prof. Ülo Langel’s group in 2011²⁹. Its sequence is the result of several modifications of the sequence of transportan. Transportan was designed in 1998 and is a chimeric CPP obtained after fusing the N-terminal part of the neuropeptide galanin with mastoparan, a wasp venom toxin²⁴. In 2000, the study of deletion analogs of transportan led to the discovery of TP10, a twenty one amino acid CPP³⁶. TP10 was then modified with a stearic acid on its N-terminus³⁷ after Futaki et al, in 2001, demonstrated that the functionalization of polyarginine with stearic acid im- proved the delivery efficacy of oligonucleotides by one hundred-folds³⁸. The next step in the design of PepFect14 was the replacement of all the lysine residues, except one, for ornithine, in order to reduce the protease activity in the cells. Indeed, ornithine being a non-proteogenic amino acid, it is less prone to be recognized by the active site of intracellular proteases. Furthermore, every isoleucine was changed for a leucine in order to decrease steric repulsion that may affect the affinity for complexed cargoes and a charge reorganization

was called PepFect14²⁹. In figure 2 are displayed the different steps of its de- sign and table 2 shows its sequence and its physico-chemical properties.

Figure 2. Steps in the design of PepFect14 from the sequence of transportan.

Table 1. Structure and characteristics of PepFect14. The net charge of the peptide is given at physiologic pH. (Stearyl: stearic acid; NH2: amidated C-terminal).

Name Structure Length MW

(g.mol^-1)

Net charge PepFect 14 Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2 21 aa 2518 +5

Thanks to its design PepFect14 is highly resistant to proteolytic activities and is not degraded before achieving transfection. It also is able to form com- plexes with a wide range of genetic materials, from antisense oligonucleotide to plasmid DNA, and typically forms particles with diameters in the range of 10² nm in cell culture media^29,39,40. These particles show a positive z-potential in milliQ water where the complexes are formed⁴¹ but this potential becomes negative in the presence of serum⁴², probably due to the presence of counter anions like albumin. PepFect14 is able to bind and deliver splice correcting oligonucleotides, small interfering RNA as well as plasmid DNA29,40,43,44. In particular, PepFect14 showed a remarkable efficacy for the delivery of splice correcting oligonucleotide into HeLa pLuc705 cells, mdx mouse myotubes and a Duchenne’s muscular dystrophy in vitro model⁴⁰.

Uptake mechanisms and cellular trafficking

The first studies on the uptake mechanisms used by CPPs to penetrate cells pointed toward direct translocation through the membrane via destabilization of the phospholipid bilayer^24,45–47. Indeed, electrostatic interactions and hydro- gen bond formations can induce pore formation in the plasma membrane that lead to the entry of the peptide. A summary of the different model of pore formation is given in figure 3. This uptake mechanism is preferred by highly charged CPP like polyarginines used at high concentration. The osmotic un- balance created by high concentration of CPPs increases the membrane desta- bilization.

Figure 3. Different models of direct translocation through plasma membrane. In the barrel stave pathway, hydrophobic domain of the CPP interacts with the lipidic chains of the phospholipids, opening wells in the structure of the membrane and allowing through which CPPs can enter into the cytosol. In the toroidal pore pathway, hydro- philic interactions between the polar heads of the phospholipids and the charged resi- dues of the peptide chain also open pores in the membrane. The carpet model de- scribes the embedment of amphipathic peptides in the bilayer membrane that in turns disrupts the membrane in a detergent-like fashion creating micelles of phospholipids.

This last model is followed by antimicrobial peptides as the disruption of the mem- brane induce toxic effects. Reprinted with permission⁴⁸.

More recent studies showed that, in the case of a large cargo delivery, direct translocation is not the most used uptake mechanism but endocytosis path- ways play a more important role^49–51. Endocytosis is a fine tuned process that is not yet entirely understood. The overall principle is the engulfment of ex- tracellular material into vesicles that follow different possible intracellular trafficking pathways. The word endocytosis was first used in 1963 by Chris- tian De Duve⁵² but the significance of phagocytosis, a type of endocytosis, was already reported by Ilya Ilitch Metchnikov in the 19th century⁵³. Several

type of endocytosis have been described since then, and the three major path- ways of internalization in intracellular vesicles are the clathrin-mediated en- docytosis (CME), the caveolae-dependent endocytosis and the macropinocy- tosis⁵⁴. Clathrin/caveolae-independent endocytosis pathways have also been reported but very little information is known about it. Endocytosis is nowa- days considered as the main mechanism of uptake for CPPs. Figure 4 shows a schematic representation of these three endocytic pathways and the trafficking of intracellular vesicles.

Defining an exact uptake mechanism for a certain CPP is probably an un- reachable goal and there are many conflicting results in the literature. It is now accepted by the scientific community that CPPs can use various uptake mech- anisms to enter cells, sometimes simultaneously. Prof. Brock’s group showed in 2007 that Antp, TAT and nona-arginine were taken up simultaneously through macropinocytosis, clathrin-mediated endocytosis and caveolae-de- pendent endocytosis⁵⁵. The mechanism of entry can also differ depending on other experimental settings such as the cell line, the concentration of the treat- ment, the nature and size of the cargo and even the binding method of the cargo to the CPP^48,56.

Once a CPP and its cargo have been taken up into endosomes, another key point in achieving an intracellular therapeutic effect is endosomal escape. In- deed, the endosomal trafficking usually leads to lysosomal degradation and cellular recycling functions. Whereas, for the cargo to induce a biological ef- fect, it needs to be delivered to specific cellular compartment such as the nu- cleus for plasmid DNA or splice correcting oligonucleotide, the cytoplasm for interfering RNA and therapeutic proteins or, in some cases, mitochondria. En- dosomal escape is considered as the limiting factor in CPP transfection effi- cacy^57,58 and several chemical strategy have been used in vitro to assist this process. Most of these strategies are based in osmotic swelling that disrupt endosomes. Among them can be found chloroquine⁵⁹, sucrose⁶⁰ and calcium

Figure 4. Endocytosis and intracellular trafficking of endosomes. The uptake of CPPs and their cargoes are followed by intracellular processes leading to early endosome.

These early endosomes follow their own cellular trafficking toward late endosome and lysosome by lowering their pH but can also lead to multivesicular bodies and exocytosis. In this case, exosomes are release in the extracellular environment and can be re-distributed to neighboring cells or the same cell through a reuptake.

As for most CPPs, PepFect14’s uptake mechanism is as of today still not entirely defined but it has been shown that endocytosis is mainly used by Pep- Fect14 in complex with large cargoes such as oligonucleotides (PF14:ON)²⁹. Furthermore, Ezzat et al proved the involvement of scavenger receptor type A (SCARA) in the uptake of PF14:ON⁶². Indeed, pharmacological inhibitors of SCARA entirely abolished the splice correction induced by PepFect14 in com- plex with a splice correcting oligonucleotide (PF14:SCO). This effect was fur- ther confirmed by a siRNA knockdown of SCARA type 3 and 5 that reduced

the biological activity of PF14:SCO by 50% whereas the overexpression of the receptors increased the transfection of plasmid DNA assisted by Pep- Fect14. A follow up study by Lindberg et al showed the same effect of SCARA knockdown on the transfection assisted by a D-amino acids version of PepFect14 indicating that the effect was not mediated by an amino acid dependent interaction⁶³.

SCARA is a receptor known to recognize and/or to participate in the endo- cytosis of nucleic acids and polyanionic molecules^64,65. PepFect14 is posi- tively charged at physiological pH but the surface potential of the nanoparti- cles formed by PF14:ON in presence of serum is negative due to counter ions in solution. Juks et al. showed that SCARAs are not always present at the cell surface, but stored in vesicles and recruited from the cytoplasm upon interac- tions of PepFect14 with the cell membrane indicating an intracellular signal- ing regulating the process⁶⁶. The recruitment of SCARA led to the uptake of PF14:ON via macropinocytosis and caveolae-dependent endocytosis. Another study from Prof. Pooga’s group showed the involvement of many other factors in the uptake of PF14:ON such as intra- and extracellular calcium ions, the actin cytoskeleton, PI3K kinase and the presence of serum in the cell culture media⁶⁷. The mechanisms behind the uptake of PepFect14 are still only par- tially known and could be dependent on many different factors, from unspe- cific hydrophobic interactions with the cell membrane to specific intracellular signaling pathways leading to the recruitment of cell surface receptors.

Signaling Pathways

In cells information is carried via signaling pathways. A molecular event, such as a chemical interaction on the membrane, a change in temperature or the activation of transcription factors, is transduced throughout the cell via a

surface can lead to an entire chain of intracellular chemical reactions and phys- ical interactions allowing the cell to appropriately adapt to its extracellular environment⁶⁹. Therefore, when a CPP interacts at the cell surface, potential intracellular signaling pathways are triggered. For example, PepFect14 inter- acts with the plasma membrane and triggers an intracellular signaling cascade that recruits SCARA to the cell surface^63,66,67. In turn, SCARA induces a sig- naling pathway that promotes the recruitment of clathrin for CME and the ac- tin filament reorganization that leads to macropinocytosis. Investigating the signaling pathways modulation induced by the entry of CPP is a promising way to improve the efficacy of CPPs. This approach is more global than the study of cell-surface receptors and gives an insight on the intracellular traf- ficking of CPPs in addition to the possible interactions at the plasma mem- brane. Studying signaling pathways is not restricted to CPPs and can be used for other delivery systems. The popularization of techniques such as RNA se- quencing and high-throughput assays offer a great opportunity to understand how cells react to transfection events.

Autophagy

Cells possess their own intracellular recycling system called autophagy.

The etymology of the word comes from ancient Greek and literally translate to English as “self-eating”. Autophagy is the regulated degradation of intra- cellular components such as unfunctional proteins or ineffective organelles to provide a nutrient regeneration⁷⁰. In case of cell starvation, the autophagic pathways of nutrient regeneration are particularly stimulated to support the vital production of adenosine triphosphate (ATP)⁷¹. Even though autophagy is a degradative process, it is also a cytoprotective system, notably in cases of neurodegenerescence⁷². However, autophagy has a bivalent role in the devel- opment of tumors and can either promote or inhibit tumor growth. Pharmaco- logic interferences in the signaling pathways of autophagy is a promising field

in drug discovery. Figure 5 presents the therapeutic strategy targeting au- tophagic signaling pathways.

Figure 5. Pharmacological actions on autophagic signaling pathways. Reprinted with permission under the Creative Commons Attribution-NonCommercial-No Deriva- tives License⁷².

Autophagy is subdivided into three categories, depending on how the tar- geted material is delivered to autophagolysosomes. First, in microautophagy the membrane of a lysosome elongates and engulfs a cell component for fur- ther degradation⁷³. The second category, for which a specific organelle called autophagosome is dedicated, is called macroautophagy. The membrane of a phagosome elongates to sequester the targeted material in a double membrane autophagosome. The autophagosome, through intracellular trafficking fuses

The last category of autophagy is the chaperone-mediated autophagy (CMA).

During CMA the chaperone protein HSC70, a member of the heat shock pro- tein 70kDa (HSP70) family, recognizes the amino acid sequence KFERQ in target proteins⁷⁵. The substrate binding domain (SBD) of HSC70 binds to KFERQ motifs causing the HSC70-protein complex to move toward the mem- brane of lysosomes where the protein LAMP-2A assists the lysosomal uptake of the complex for further recycling⁷⁶. CMA is a regulation system of pro- teasomal degradation and controls the cellular proteome balance. A descrip- tion of these three categories of autophagic processes are displayed in figure 6.

Figure 6. Schematic representation of the three categories of autophagic pathways.

Reprinted from with permission⁷⁷.

Delivery of therapeutics

The intracellular insertion of therapeutics in living cells is a process that can be divided in two categories: stable and transient transfection. A stable transfection requires the integration of genetic material into the genomic DNA. The coded protein can then have a sustained expression because the DNA is replicated during cell division. A transient transfection relates to the insertion of therapeutics into the cell but without integration in the genome. In this case different type of therapeutics can be used: RNA, DNA or protein.

The transient delivery of RNA and DNA will lead to a time-limited expression or silencing of a protein⁷⁸. The insertion of an already formed protein leads to biological function and is also limited in time due to the stability and turnover of the protein. These two possible outcomes of the delivery of therapeutics are illustrated in figure 7.

Figure 7. Illustration of a stable transfection (A) and a transient transfection (B) of therapeutics into a cell. Adapted and reprinted with permission under the Creative Commons license (Attribution-Noncommercial)⁷⁸.

Gene therapy

In 1967, fifteen years after the capture of X-ray diffraction images of DNA by Rosalind Franklin⁷⁹, Joshua Lederberg and Edward Tatum introduced the concept of gene therapy during a symposium called “Reflection on Research and the Future of Medicine”⁸⁰. Ethical considerations rapidly arose, and a few years later, Theodore Friedmann and Richard Roblin expressed the need for a complete ethical framework⁸¹. The first clinical trial of gene therapy on a hu- man was subsequently performed in the early 1990s⁸².

The principle of gene therapy is the treatment of cells with genetic material with the therapeutic aim to alter the gene expression. It is achieved by the insertion of oligonucleotides into the transcription machinery and can lead to the silencing, correction, alteration, deletion or addition of targeted genes and can even combine those effects depending on the nature of the inserted genetic material⁸³. Gene deletion interferes directly with genomic DNA and thus in- duces a permanent silencing that suppress the expression of a non-functional or disease-related protein. The addition of a new gene can be either stable or transient depending on the integration in the genome, and aims to supply a protein that is not naturally expressed. An example of stable gene addition is given by mammalian cell lines stably expressing firefly luciferase⁸⁴. Aberrant mutations of a gene also lead to unfunctional proteins and can be treated by gene correction or alteration. This can be achieved through action on splicing events⁸⁵. Acting on splicing event provides a transient transfection and is therefore less invasive than a gene deletion or addition. A specific gene can be silenced through RNA interferences where messenger RNA (mRNA) is sequestered from transcription without compromising the genomic DNA⁸⁶. This technique induces a transient knockdown of a protein expression as the interfering RNA is eventually degraded⁸⁷.

Nowadays, there are many available tools to perform gene therapy both in

vectors. Viral vectors have been extensively used since 1990s to transfect genes⁸⁹. The use of viral vectors in gene therapy is not surprising as viruses are natural agents of gene transfection and, therefore, are highly potent. How- ever, evolution has provided organisms with natural defenses against virus in- vasion. These defenses act as barriers to viral vector as they induce side effects such as immunogenicity, allergic reactions, host rejection, mutagenicity and oncogenicity⁹⁰. Non-viral vectors are a large class of delivery systems that encompasses polymers, cationic lipids, nanoparticles and, of course, CPPs.

Non-viral vectors usually induce a minimal host immune response and can achieve cellular targeting. They are suitable for localized and topical treatment to target tissues⁹¹. Recently a very promising technology called CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9) emerged⁹². With CRISPR/Cas9, an intracellular sequence specific editing of DNA can be achieved with a technique that can be compared to a cut-and-paste method⁹³.

Gene therapy is a highly potent tool that can be used for the treatment of genetic diseases such as Parkinson’s disease⁹⁴, Alzheimer’s disease⁹⁵, muscu- lar dystrophy⁹⁶ or type 1 diabetes⁹⁷.

Antisense therapy

An antisense therapy is a gene therapy that relies on the transfection of antisense oligonucleotides (ASO). An ASO is a complementary sequence to a native DNA or RNA sequence allowing it to bind to the target nucleotides sequence. Two strategies of antisense therapy can be employed depending on the target sequence. If the antisense oligonucleotide is complementary to a genomic DNA sequence, upon binding to the target the transcription will be prevented stopping the production of mRNA and the expression of the protein.

This strategy is also called antigene therapy⁹⁸. Another possible target of an- tisense therapy is mRNA, in which case the expression of the protein will also be inhibited as a complex between mRNA and an antisense oligonucleotide recruits ribonucleases H (RNase H) that degrades mRNA without translation.

RNase H binds to double-stranded RNA and degrades both strands. This fea- ture is a disadvantage as the degradation of the antisense oligonucleotide avoids reaching an enzymatic kinetic of the knockdown⁹⁹

The discovery of pre-mRNA splicing in 1977¹⁰⁰ offered new opportunities for antisense therapy¹⁰¹. It was shown that genes are actually a composite of coding exons and non-coding introns^102,103. During the splicing process, the pre-mRNA’s sequence, which is a complementary strand of an entire gene, is cleaved into exonic and intronic parts and after removal of the non-coding introns, the exons are joined together to give mRNA. This junction process is competitive and, therefore, several alternative splices result¹⁰⁴. By alternative splicing, a single gene can be transcribed into upwards of ten thousands vari- ant proteins¹⁰⁵. The splicing process is an important factor of the proteomic diversity, but a disfunction in the splicing machinery can lead to aberrant splicing. Many neurological diseases such as neurofibromatosis type 1, spinal muscular atrophy, schizophrenia, autism spectrum disorders and myotonic dystrophy 1 and 2 among others, have been linked to aberrant splicing¹⁰⁵. Splice correcting oligonucleotides (SCO) are a tool to re-orient the splicing process and a subtype of ASOs. Indeed, their sequence is complementary to a specific region at the end or beginning of introns. Upon binding to the target intronic region, the splice machinery is redirected to either include or exclude exons in mRNA. The transfection of SCOs is then able to correct aberrant splicing which gives it a high potential as a therapeutic agent.

Targeted delivery to intracellular organelles

To induce a biological and therapeutic effects drugs usually have to be de- livered to a specific cellular component. For example, a stable transfection requires the integration of new genetic material into the genome and, there-

The same applies for the delivery of SCO as the splicing of pre-mRNA takes place in the nucleus^108,109. To the contrary, small interfering RNAs that inter- fere with mRNA can be delivered in the cytoplasm and do not require a nu- clear localization¹¹⁰. Intracellular targeted delivery is, however, not limited to the cytosol and nucleus. In the literature, multiple examples of peptides tar- geting subcellular components can be found. Lysosomal and endosomal tar- geting has been achieved by HIV-1 TAT, fibrinogen-derived ICAM-1 binding sequence and lysosome targeting peptide^111–113. The endoplasmic reticulum (ER) has been shown to be targeted by ER-insertion signal¹¹⁴ or ER targeting moieties – for example AAKKAA¹¹⁵.

Mitochondria as a therapeutic target

Mitochondria are considered as the powerplant of cells. They are the cell component responsible for the vital production of ATP through a pathway called the oxidative phosphorylation (OXPHOS). There are several hypothe- ses regarding their origin in eukaryote cells, but the most commonly accepted is the endosymbiotic theory. In this hypothesis, mitochondria originate from an evolutionary symbiosis between an eukaryote cell and an aerobic prokary- ote organism^116,117. Through engulfment, the prokaryote cell gained the pro- tection offered by the environment of the cytoplasm of the host cell while the host cell relied on the engulfed cell for energy production. Through evolution, the prokaryote cell became the mitochondria.

Mitochondria are unique organelles that possess their own independent DNA (mtDNA)¹¹⁸. Their structure is composed of the outer mitochondrial mem- brane (OMM) separated to the inner mitochondrial membrane (IMM) by the intermembrane space. The inside of the IMM is called the matrix and the ridge structures that can be observed with electron tomography are named cristae mitochondriales. The cristae are of major importance for OXPHOS. The entry in mitochondria is regulated by different proteins. The translocase outer mem- brane (TOM)¹¹⁹ and the voltage-dependent anion channel (VDAC)¹²⁰ regulate

the entry in the intermembrane space¹²¹ while the translocase inner membrane (TIM) discriminates for the passage in or out of the matrix^122,123. The OXPHOS pathway, through the respiratory chain, generates a mitochondrial membrane potential (DYm) that normally ranges between 80 and 140 mV. A DYm above 140 mV has been reported to displace the production of ATP to a production of reactive oxygen species (ROS)¹²⁴. Mitochondria also play a role in the regulation of intracellular calcium concentration and cell death signal- ing. The vital processes that involve mitochondrial activity make mitochon- drial dysfunctions a critical component of many diseases. Therefore, the tar- geted delivery of therapeutics to mitochondria holds tantalizing potential in modern therapies. These therapies are so far limited by the difficult penetra- tion of mitochondrial membranes.

MtCPP1, a CPP developed by Cerrato et al. in 2015, has been shown to be able to target mitochondria¹³. Additionally, mtCPP1 has the intrinsic ability to reduce the level of ROS. However, the short sequence of mtCPP1 composed by four amino acids only does not allow for the binding and delivery of oligo- nucleotides.

Protein delivery

An alternative to gene therapy is the delivery of therapeutic proteins. In- deed, gene therapy is based on the insertion of genetic material in order to produce a missing protein or to replace a dysfunctional protein. A viable strat- egy is then to directly deliver the protein of interest. This strategy presents several advantages¹²⁵. First, the direct introduction of a protein is an elegant way of bypassing the transcription factors regulating gene expression. The transcription and translation from gene to protein is a complex process that involves many uncontrollable factors. Moreover, the insertion of a new gene

the splicing of the imported gene can also become a limiting factor as many variant protein can be expressed from a single gene¹⁰⁴. Second, the delivery of therapeutic proteins allows for a control of the time frame of a treatment. In- deed, a delivered protein is immediately available for a biological action. Fur- thermore, the use of therapeutic proteins brings more safety and less ethical considerations than the insertion of genetic material. The natural degradation of proteins by proteases results in transient therapies that allows for the ability to control the dosage of a treatment over time. The limiting factor in the de- velopment of protein-based therapy is the same as for gene therapy, namely the delivery. Proteins are large and charged molecules and therefore are often unable to cross cell membranes. The use of proteins for therapy is then closely linked to the development of a carrier that can transport them to specific in- tracellular sites^7,8,126. To summarize, the delivery of protein offers a high po- tency, an increased safety and more control over a treatment than gene ther- apy¹²⁷.

Heat shock protein 70kDa

Heat Shock Protein 70kDa (HSP70) are a family of proteins involved in many cellular processes. HSP70s are highly conserved among eukaryotes and prokaryotes and all the members of this family are proteins with a molecular weight around 70 kDa that share the same chaperone properties based on three domains with distinct functions¹²⁸. The substrate binding domain (SBD) is able to bind to hydrophobic polypeptide sequences. The binding affinity is modulated via the adenine nucleotide binding domain (NBD) on the N-termi- nal¹²⁹. ATP binding to the NBD yields in a low affinity for the substrates and thus a fast turnover of the binding events. Upon binding of adenosine diphos- phate (ADP), the a-helical C-terminal acts as a lid and close the SBD, there- fore strengthening the affinity of binding and locking the substrate in the rigid structure of the SBD¹³⁰. HSP70s are considered as protective proteins and have

a major role in the folding process of nascent protein^131,132, refolding of dys- functional proteins¹³³, protection against external stress such as chemical or physical stress^134,135 and also regulate protein levels via modulation of tran- scription factors¹³⁶ and degradation of proteins¹³⁷. These roles can be divided in two categories: the processing of misfolded proteins and the protection against stress-induced aggregation (Figure 8).

Figure 8. A) Role of HSP70 in the processing of misfolded proteins. B) The mecha- nism of HSP70-mediated protection against stress-induced aggregation.

therapeutic cargo to be delivered in cells. Its protection function against pro- tein aggregation has the potential to play an important role in therapies against aggregation of a-synuclein in Parkinson’s disease¹³⁸ or amyloid-b and tau in Alzheimer’s disease¹³⁹.

A ^ims

This thesis focused on the pre-existing cell-penetrating peptide PepFect14.

The experimental work presented here was directed toward two main objec- tives:

• To provide a better understanding of the intracellular signaling of the cell-penetrating peptide PepFect14 in order to improve its transfection efficacy.

• To develop new applications for PepFect14-assisted transfections in order to expand its potential as a therapeutic delivery system.

The aims for each paper are detailed on the next pages.

Paper I

The aim of paper I was to use a library of small molecule drugs to modulate signaling pathways in order to increase the efficacy of the transfection of oli- gonucleotides in complex with PepFect14. The secondary aim of this paper was to identify the signaling pathways that produced an effect on the transfec- tion and to link them to PepFect14 uptake mechanism.

Paper II

The aim of paper II was to determine the intrinsic effects of PepFect14, alone or in complex with oligonucleotides, on intracellular signaling by using a whole-transcriptome analysis. Additionally, we aimed at unravel the signal- ing pathways involved in PepFect14 intracellular trafficking and to use this knowledge to modulate PepFect14 transfection efficacy.

Paper III

Findings from paper II suggested that PepFect14 and HSP70 were able to form a complex together. Based on these results, paper III aimed at verifying this possible interaction and to use it to achieve the first protein delivery using PepFect14. HSP70 being a major protective protein in virtually all cells, its delivery aimed at major advances in protein misfolding and aggregation re- lated diseases.

Paper IV

In paper IV, we investigated the potential of several formulation of Pep- Fect14 and mtCPP1 to combine the property of both CPPs to achieve an or- ganelle targeted gene therapy. mtCPP1 has the ability to translocate into mi- tochondria while PepFect14 can mediates the transfection of oligonucleotides.

The combination of these two properties aimed at delivering an antisense oli- gonucleotide to mitochondria and to regulate the expression of mitochondrial proteins. There are numerous diseases that originate from mitochondrial dis- function and the regulation of mitochondrial DNA transcription has the poten- tial to reverse or alleviate these dysfunctions.

M ^ethods

A detailed description of each method used can be found in the methodo- logical section of the papers included in this thesis. This section presents a theoretical background of the methods.

Solid-Phase Peptide Synthesis

Solid-Phase Peptide Synthesis (SPPS) is a method for the synthesis of pep- tides that is based on orthogonal protection groups and cycles of coupling, washing and deprotection where the amino acids are coupled one after the other from the C-terminal to the N-terminal of the sequence. It was first de- veloped at the end of the 1950s and beginning of the 1960s. Robert Bruce Merrifield published the method in 1963¹⁴⁰ and got awarded with a Nobel Prize in chemistry in 1984. The first amino acid of the sequence is covalently bound on its carboxylic acid to a solid support that acts as a permanent pro- tection while its a-amine group is orthogonally protected. The a-amine group can be selectively deprotected to allow the coupling of a new protected amino acid that will, in turn, be deprotected to allow the next coupling. The solid support - also called resin - is insoluble in the reaction solvent. This feature allows simple washing steps as the peptide chain can be rinsed at every step while being contained in a reaction vessel closed with a filter. The side chain of amino acids can also be orthogonally protected, meaning that side reactions are avoided and that any functionalization of the peptide chain can be regio-

selectively achieved. The simplicity of this method combined with its ele- gance and high yield makes SPPS the most commonly used peptide synthesis method^141,142. When the peptide chain is synthesized, it can be cleaved from the resin and extracted in organic solvent such as diethyl-ether or chloroform.

All the peptides used in this thesis were synthesized using 9-fluorenylmethox- ycarbonyl (Fmoc) based SPPS (Figure 9). In Fmoc-based SPPS, the a-amine group of each amino acid is protected with Fmoc and can be deprotected by the action of a mild base. The resin can only be cleaved by the use a strong acid and the side chain of amino acids can be protected by either a strong acid- labile protection or a mild acid-labile protection. In the latter, the selective functionalization of the side chain is allowed by the method.

Figure 9. Iterative process used in Fmoc-based solid phase peptide synthesis.

All the peptides used in this thesis were synthesized using a fully automated microwave peptide synthesizer (Alstra+, Biotage AB, Uppsala, Sweden) with

Fmoc groups were removed by the action of 20% piperidin in dimethylforma- mide and the carboxylic acids were activated with N,N'-diisopropylcar- bodiimide (DIC) and Oxyma Pure (Figure 10). Side chains that required func- tionalization were protected with the mild acid-labile 4-methyltrityl which can be selectively removed by a 1% trifluoroacetic acid (TFA) solution. The stea- ric acid was coupled using the same protocol as for amino acids.

Figure 10. Schematic representation of the activation of a carboxylic acid and cou- pling assisted by DIC and Oxyma Pure.

The peptide chain was cleaved from the resin with a 95% TFA, 2.5% triiso- propylsilane and 2.5% ultra-pure water solution yielding in a solution contain- ing the peptide, deletion analogues of the peptide, scavengers and protective groups. The peptidic compounds were extracted by precipitation in cold di- ethyl ether, dissolved in a mix of acetonitrile and ultra-pure water (respec- tively 80% and 20%) containing 0.1% TFA before purification in a semi pre- parative reversed phase high performance liquid chromatography (RP- HPLC). The purified fractions were then analyzed using ultra-high perfor- mance liquid chromatography mass spectrometry (UHPLC-MS) or elec- trospray ionization time of flight mass spectrometry (ESI-TOF). The purified peptides were then freeze-dried and reconstituted in ultra-pure water before being stored at -20°C. All the peptide used in this thesis are listed in Table 3.

Table 3. List of the peptides used in this thesis together with their sequence.

Stearyl: stearic acid; r: D-arginine; Y^a: 2,6-L-dimethyltyrosine; O: L-ornithine;

NH2: amidated C-terminal.

Name Sequence

PepFect14 Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2 mtCPP1 rY^aOF-NH2

mitFect1 AGYLLGK(Stearyl-𝜺N)LLOOLAAAALOOLL-NH2 mitFect2 rY^aOFAGYLLGK(Stearyl-𝜺N)LLOOLAAAALOOLL-NH2 mitFect3 Stearyl-AGYLLGK(rY^aOF-𝜺N)LLOOLAAAALOOLL-NH2 mitFect4 Stearyl-rY^aOFAGYLLGKLLOOLAAAALOOLL-NH2 mitFect5 rY^aOFAGYLLGKLLOOLAAAALOOLL-NH2

mitFect6 AGYLLGK(rY^aOF-𝜺N)LLOOLAAAALOOLL-NH2

mitFect7 AGYLLGK(Stearyl-rY^aOF-𝜺N)LLOOLAAAALOOLL-NH2 mitFect8 Stearyl-AGYLLGKLLOOLAAAALOOLLrY^aOF-NH2 mitFect9 Stearyl- rY^aOF-NH2

Cell cultures

In Paper I, II and IV HeLa pLuc 705 were used. HeLa cells were first har- vested from a cervical cancer biopsy in 1951. The name of the cells comes from the patient from whom they were harvested, Henrietta Lacks. The cell line was first propagated by George Otto Gey¹⁴³. HeLa cells are the first hu- man immortal cell line established for in vitro cell culture and are now widely used for research in cell biology. The history of this cell line can be found in the book The Immortal Life of Henrietta Lacks¹⁴⁴ which has recently been adapted into a movie in 2017. The cell line HeLa pLuc 705 was constructed from HeLa cells that were stably transfected with a luciferase-encoding gene interrupted by a mutated b-globin intron. This intron contains an aberrant splice site at the nucleotide 705 yielding in a wrongly spliced and unfunctional luciferase protein¹⁴⁵. The advantages that this wrong splicing procures will be presented in the next section.

The cell line that was used in paper III is called Bomirski Hamster Mela- noma pLuc (BHM pLuc). BHM cells were first isolated in 1959 from a spon- taneous melanoma in a Syrian golden hamster and have been maintained through transplantation in various animals. These cells were melanotic mela- noma and are usually referred to as BHM Ma cells. By spontaneous alteration, an amelanotic cell line originated from the BHM Ma in 1963 and are called BHM Ab cells. BHM Ab cells were found unable to produce melanin, showed an increased growth rate and lost the ability to metastasize^146,147. The BHM pLuc cells that was used in paper III are BHM Ab cells that have been stably transfected with a luciferase-encoding gene and, thus, stably expressed a func- tional luciferase protein.

Both cell lines were cultivated at 37,5°C, 5% CO2, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glutamax, 0,1 mM non-essen- tial amino acids, fetal bovine serum (FBS), Plasmocyn (5 µg/mL) and the an- tibiotics penicillin (200 U/mL) and streptomycin (200 µg/mL).

The treatment protocol for every method used in the building of this thesis can be found in the corresponding section of the four papers.

Luciferase assay

The glow that can be seen in fireflies and click beetles is due to an enzy- matic reaction catalyzed by luciferase. Indeed luciferase catalyzes the oxida- tion of luciferin into oxyluciferin in presence of ATP and oxygen and this reaction produces a photon making these insects glow in the dark (Figure 11).

This phenomenon is known as bioluminescence. Since bioluminescence is an easy feature to measure, luciferase has become one of the most commonly used reporter gene¹⁴⁸. Its sensibility and linear response range made it a suita- ble reporter enzyme for the quantification of gene expression. For example the transfection of a luciferase plasmid into cells that do not express luciferase can be monitored with a measurable increase in light emission of a cell lysate mixed with luciferin and other cofactor¹⁴⁹. In the same way, the successful transfection of an siRNA against luciferase into cells that express luciferase will result in a decreased light emission¹⁵⁰. The luciferase reporter can also be used to report the action of chaperone protein involved in protein folding as a misfolded luciferase will not catalyze the oxidation of luciferin¹⁵¹.

Figure 11. Luciferase-assisted oxidation of luciferin yielding in bioluminescence.

Splice correcting assay

The luciferase splice correcting assay that was used in paper I, II and IV was first developed in Ryszard Kole group and is described in Kang et al (1998)¹⁴⁵. In this assay, HeLa pLuc 705 cells are used to assess the delivery of SCOs. In HeLa pLuc 705 cells, a gene encoding for luciferase is interrupted by a mutated intron yields in a wrongly spliced and non-functional luciferase protein. A successfully delivered SCO hides the cryptic splicing site and re-

orient the splice machinery toward the production of a functional luciferase.

Upon lysis of the cells and addition of D-luciferin together with co-factors, the luciferase-induced light emission can be quantified in a luminometer (Fig- ure 12). This light emission is directly correlated to the amount of functional luciferase and, thus, to the efficacy of the splice correction. In paper I, II and IV, we used a 2’OMePS antisense oligonucleotide (Odense, Denmark) to per- form the splice correction.

Figure 12. Splice-correction reporter assay in HeLa pLuc 705. Reprinted with per- mission⁷⁷.